Alkyne-tagged SERS nanoprobe for understanding Cu+ and Cu2+ conversion in cuproptosis processes

Simultaneously quantifying mitochondrial Cu+ and Cu2+ levels is crucial for evaluating the molecular mechanisms of copper accumulation-involved pathological processes. Here, a series of molecules containing various diacetylene derivatives as Raman reporters are designed and synthesized, and the alkyne-tagged SERS probe is created for determination Cu+ and Cu2+ with high selectivity and sensitivity. The developed SERS probe generates well-separated distinguishable Raman fingerprint peaks with built-in corrections in the cellular silent region, resulting in accurate quantification of Cu+ and Cu2+. The present probe demonstrates high tempo-spatial resolution for real-time imaging and simultaneously quantifying mitochondrial Cu+ and Cu2+ with long-term stability benefiting from the probe assembly with designed Au-C≡C groups. Using this powerful tool, it is found that mitochondrial Cu+ and Cu2+ increase during ischemia are associated with breakdown of proteins containing copper as well as conversion of Cu+ and Cu2+. Meanwhile, we observe that parts of Cu+ and Cu2+ are transported out of neurons by ATPase. More importantly, cuproptosis in neurons is found including the oxidative stress process caused by the conversion of Cu+ to Cu2+, which dominates at the early stage (<9 h), and subsequent proteotoxic stress. Both oxidative and proteotoxic stresses contribute to neuronal death.


Supplementary Discussion
Calculation of enhancement factor.
A single particle containing ~ 10 sharp tips branching out from a central spherical core, and a normally distributed branch length of 14.50±4.40nm was considered with a width of 10.10±2.40nm at the base and a tip apex diameter of 4.90±1.10nm, and the core diameter was fixed at 20.50±3.40 nm.Thus, the surface area AGNs of the individual particle was calculated to be ~4744.04nm 2 , and GNs were mixed with an excess of EETP, and resuspended by centrifugation to obtain GNs with the surface maximally modified by EETP (0.5 mg mL -1 ).Nads was the number of EETP in the excitation volume for GNs colloids.The excitation volume was calculated by using the equation: Vlens = π(d/2) 2 H, where d was the diameter of the beam size (d = 3.83 µm), H was the effective depth of focus (H = 22 µm), the effective excitation volume was 253.46 μm 3 for Raman microscopy with 785 nm excitation using the 10× objective.ρads,MBN was the bonding density of EETP onto GNs surface as reported (~49.89±9.12pmol cm -2 ). 31 We calculated: Nads = ~4.35×10 5 , Ibulk = 123.28,ISERS, GNs = 917.27.EF was estimated to be (7.42±0.63)× 10 6 .

Table 2 .
Raman bands assignment of Cu 1 R2 before and after addition of Cu + .

Table 3 .
Raman bands assignment of Cu 1 R3 before and after addition of Cu + .

Table 4 .
Raman bands assignment of Cu 1 R4 before and after addition of Cu +

Table 5 .
Raman bands assignment of Cu 1 R5 before and after addition of Cu + .

Table 6 .
Raman bands assignment of Cu 2 R1 before and after addition of Cu 2+ .

Table 7 .
Raman bands assignment of Cu 2 R2 before and after addition of Cu 2+ .

Table 8 .
Raman bands assignment of Cu 2 R3 before and after addition of Cu 2+ .

Table 9 .
Raman bands assignment of Cu 2 R4 before and after addition of Cu 2+ .